Detection of erythropoietin and erythropoietin receptor

ABSTRACT

Methods and kits for simultaneous measurement of erythropoietin and erythropoietin receptor in a biological sample are described.

TECHNICAL FIELD

[0001] This invention relates to assays for simultaneous detection oferythropoietin (Epo) and erythropoietin-receptor (EpoR). In particular,immunoassays and PCR-based assays are described for simultaneouslydetecting Epo and EpoR, and related nucleic acids.

BACKGROUND OF THE INVENTION

[0002] Erythropoietin (Epo) is a glycoprotein hormone of molecularweight 34 kilodaltons (kDa) that is produced in the mammalian kidney andliver. Epo is a key component in erythropoiesis, inducing theproliferation and differentiation of red cell progenitors. Epo activityalso is associated with the activation of a number of erythroid-specificgenes, including globin and carbonic anhydrase. Bondurant et al., Mol.Cell Biol. 5: 675-683 (1985); Koury et al., J. Cell. Physiol. 126:259-265 (1986). The erythropoietin receptor (EpoR) is a member of thehematopoietic/cytokine/growth factor receptor family, which includesseveral other growth factor receptors, such as the interleukin (IL)-3,-4 and -6 receptors, the granulocyte macrophage colony-stimulatingfactor (GM-CSF) receptor as well as the prolactin and growth hormonereceptors. Bazan, Proc. Natl. Acad. Sci USA 87: 6934-6938 (1990).Members of the cytokine receptor family contain four conserved cysteineresidues and a tryptophan-serine-X-tryptophan-serine motif positionedjust outside the transmembrane region. The conserved sequences arethought to be involved in protein-protein interactions. Chiba et al.,Biochim. Biophys. Res. Comm. 184: 485-490 (1992).

[0003] EpoR cDNA has been isolated recently from mouse liver (Tojo etal., Biochem. Biophys. Res. Comm. 148: 443-48 (1987)) and from humanfetal liver. Jones et al., Blood 76: 31-35 (1990); Winkelmann et al.,Blood 76: 24-30 (1990). The human cDNA encodes a polypeptide chain of MW˜55 kDa and having about 508 amino acids. Genomic clones of human EpoRhave been isolated and sequenced. Penny and Forget, Genomics 11: 974-80(1991); Noguchi et al., Blood 78: 2548-2556 (1991). Analysis of thecoding sequence predicts about 24 amino acid residues in a signalpeptide, about 226 amino acids in an extracellular domain, about 23amino acids in a membrane-spanning domain, and about 235 amino acids ina cytoplasmic domain. D'Andrea and Zon, J. Clin. Invest. 86: 681-687(1990); Jones et al., Blood 76: 31-35, (1990) and; Penny and Forget,Genomics 11: 974-80 (1991). The mature human EpoR protein has about 484amino acids. All human erythroid progenitor cells have been shown tocontain Epo receptors. Binding of Epo appears to decline as erythroidprogenitor cells mature, until Epo receptors are not detectable onreticulocytes. Sawada et al., J. Clin. Invest. 80: 357-366 (1987);Sawada et al., J. Cell. Physiol. 137: 337 (1988). Epo maintains thecellular viability of the erythroid progenitor cells and allows them toproceed with mitosis and differentiation. Two major erythroidprogenitors responsive to Epo are the Burst-forming units-erythroid(BFU-E) and the Colony-forming units-erythroid (CFU-E). The Epo receptornumber correlates very well with the response to Epo in normal BFU-E andCFU-E. Epo receptor numbers appear to decline after reaching the peakreceptor number at the CFU-E stage in human and murine cells. Sawada etal., J. Clin. Invest. 80: 357-366 (1987); and Landschulz et al., Blood73: 1476-1486 (1989). The recovery of Epo receptors after removal of Epoappears to be dependent on protein synthesis, which suggestsdownregulation of Epo receptor by degradation, and the subsequentupregulation of receptors by the new synthesis of receptors when Epo isremoved. Sawyer and Hankins, Blood 72: 132 (1988); and Komatsu andFujita, Cancer Res., 53:1156-1161 (1993). Studies of Epo receptors onmegakaryocytes and erythroid progenitors suggest that there is a linkbetween the regulation of erythropoiesis and thrombopoiesis, in thatstimulation of cell division by both cell types is controlled by Eporeceptor numbers. Berridge et al., Blood 72: 970-977 (1988); and Komatsuand Fujita, supra. Although the Epo receptor has been cloned, theprecise mechanisms involved in binding of Epo to Epo receptors and therelationship to subsequent erythropoietic processes are not known.

[0004] Characterization of the Epo receptor (EpoR) has been difficultdue to the extremely small quantities of EpoR that can be obtained fromnatural sources. Thus, the mechanism of Epo interaction with itsreceptor, which stimulates erythropoiesis, is still unknown. D'Andreaand Zon, J. Clin. Invest. 86: 681-687 (1990). Recently this mechanismhas been of great interest in understanding the role of growth factorsand their receptors in leukemogenesis; altered hematopoietic growthfactors and their receptors may contribute to tumorigenesis andleukemogenesis. Dunbar et al., Science 245: 1493-1496 (1989) and; Li etal., J. Virol. 57: 534-538 (1986).

[0005] Several studies of the correlation between the Epo responsivenessof a particular cell type and the affinity of the cell type for Epo havereported discordant results. These studies have used recombinant Epo orEpoR possessing some non-native amino acid sequence from thecorresponding plasmid vectors. Berridge et al., Blood 72: 970-977(1988); and Harris et al., J. Biol. Chem. 267: 15205-09 (1992). It ispossible that tertiary structural changes and/or other features of theserecombinant Epo or EpoR molecules have changed the characteristics ofthe native protein. Thus, it would be a significant advance to obtainsubstantially pure fragments of the Epo receptor, free of extraneous(e.g, vector) amino acid sequence. Although it could not be predictedwhether or not such fragments would retain functional activity,nevertheless a purified extracellular domain fragment would beparticularly useful since Epo binds to the extracellular domain of theEpo receptor.

SUMMARY OF THE INVENTION

[0006] The invention is based on the development of several sensitiveand simple methods for detecting Epo or EpoR proteins and associatedantibodies. The methods and reagents described herein represent usefultools for differential diagnosis in Epo and EpoR related clinicalproblems, as well as other hematological growth-factor-related clinicalproblems. Purification of pure human Epo-bp (Epo binding protein, i.e.,the extracellular domain of EpoR) and antibodies thereto are benchmarksthat allowed the present methods to be developed for therapeutic anddiagnostic use. Visualization of Epo-R is possible now, both in vitro inhuman samples and in vivo in animal studies. The measurement of Epo orEpoR, and antibodies thereto, will help in understanding the structuraland functional relationship in Epo/Epo-R interactions on blood cellprogenitors.

[0007] The assay method(s) described herein are able to detectnano-concentrations of Epo and EpoR-soluble proteins in the human bloodand tissue samples. Sensitive detection allows a better understanding ofthe Epo/EpoR interaction in blood cell production and related diseasesin blood cell production. Thus, the present methods allow treatmentmethods to be established to control hematological malignancies and somesystemic cardiovascular diseases.

[0008] In one aspect, the invention features a method for simultaneousmeasurement of Epo and EpoR in a biological sample. The method includesa) providing a solid substrate having first and second antibodiesattached thereto in different, discrete regions, wherein the firstantibody has specific binding affinity for Epo and the second antibodyhas specific binding affinity for EpoR, and wherein the first and secondantibodies are functional antibody fragments (e.g., Fab fragments); andb) contacting the solid substrate with the biological sample underconditions wherein Epo and EpoR in the biological sample becomes boundto the first and the second antibodies. The presence, absence, or amountof Epo and EpoR is detected on the solid substrate. The first and secondantibodies can be polyclonal antibodies.

[0009] Detecting the presence, absence, or amount of Epo and EpoR caninclude contacting the solid substrate of part b), above, with third andfourth antibodies, wherein the third antibody has specific bindingaffinity for Epo and the fourth antibody has specific binding affinityfor EpoR, and wherein the third and fourth antibodies are detectablylabeled. The third and fourth antibodies can be polyclonal antibodies.

[0010] Detecting the presence, absence, or amount of Epo and EpoR alsocan include d) contacting the solid substrate of part b), above, withthird and fourth antibodies, wherein the third antibody has specificbinding affinity for Epo and the fourth antibody has specific bindingaffinity for EpoR; and e) contacting the solid substrate of part d) withfifth labeled antibodies having specific binding affinity for the thirdand fourth antibodies. The third and fourth antibodies can be polyclonalantibodies. The fifth labeled antibodies can be labeled with an enzyme,a substrate, or a fluorescent moiety.

[0011] The invention also features a kit for detecting Epo and EpoR in abiological sample. The kit includes (a) a solid substrate (e.g.,microtiter plate) having first and second antibodies attached thereto indifferent, discrete regions, wherein the first antibody has specificbinding affinity for Epo and the second antibody has specific bindingaffinity for EpoR, and wherein the first and second antibodies arefunctional antibody fragments; (b) a first container having thirdantibodies enclosed therein, wherein the third antibodies have specificbinding affinity for Epo; and (c) a second container having fourthantibodies enclosed therein, wherein the fourth antibodies have specificbinding affinity for EpoR. The kit further can include a third containerhaving control antigen enclosed therein. The kit also can include alabel or package insert indicating that Epo and EpoR can besimultaneously detected by contacting the solid substrate with thebiological sample under conditions wherein any Epo or EpoR in thebiological sample becomes bound to the first and second antibodies andcontacting the solid substrate with Epo or EpoR bound thereto with thethird and the fourth antibodies. The first and second antibodies can bepolyclonal antibodies and can be functional antibody fragments. Thethird and fourth antibodies can be polyclonal antibodies.

[0012] The invention also features a method for detecting the presence,absence, or amount of EpoR on human blood progenitor cells. The methodincludes contacting a biological sample such as blood or bone marrowwith antibodies having specific binding affinity for EpoR, wherein thebiological sample contains human blood progenitor cells, and wherein thecontacting occurs under conditions wherein antibodies become bound toEpoR on the human blood progenitor cells in the biological sample, anddetecting the presence, absence, or amount of EpoR by identifying thehuman blood progenitor cells having antibody bound thereto. The humanblood progenitor cells can include megakaryocytes, erythroid, andmyeloid progenitor cells.

[0013] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0014]FIG. 1 is a diagrammatic representation of pJYL26, a plasmidhaving about 678 bp of the 5′ coding sequence of human erythropoietinreceptor cDNA inserted into the expression vector pGEX-2T. FIG. 1 alsodepicts the recombinant fusion protein, EpoRex-th, that is expressedfrom pJYL26.

[0015]FIG. 2A shows the absorbance at 280 nanometers (A₂₈₀) of fractionscollected from purification of an E. coli cell extract, expressingEpoRex-th, on a glutathione affinity column. FIG. 2B shows the A₂₈₀ offractions containing Epo-bp collected as a result of erythiopoietinaffinity chromatography of thrombin treated EpoRex-th.

[0016]FIG. 3 is a photograph of a Coomassie blue stained polyacrylamidegel, showing the cleavage of EpoRex-th by thrombin.

[0017]FIG. 4 is a Western blot showing binding of sheep anti-Epo-bpantibody to Epo-bp.

[0018]FIG. 5 shows the binding of various concentrations of human¹²⁵I-Epo to Epo bp, in the presence and absence of unlabeled Epo.

[0019]FIG. 6 is a photograph of a Coomassie blue stained polyacrylamidegel showing the polypeptide bands observed after trypsin digestion ofEpo-bp.

[0020] FIGS. 7A-7D are photographs of spleens isolated from rats treatedwith Epo (A-C) and from control rats (D).

[0021] FIGS. 8A-8F are photomicrographs of control bone marrow cellswith or without fluorescein labeling. FIGS. 8A-8C are of control cellsat 100×magnification (8A and 8C) or at 400×magnification (8B). FIGS.8D-8F are preimmune serum treated cells as positive controls (1000×).FIGS. 8C and 8F are fluorescein labeled, Fab-fractionated sheep serumtreated cells. It is noted that the preimmune serum treated samples (8F)did not show any receptor binding activity as the same shown on controlcells (C).

[0022] FIGS. 9A-9F are photomicrographs of fluorescein labeled receptorsites of bone marrow progenitor cells. M: Megakaryocytes;

Basophil erythroblast;

Polychromatophil erythroblast;

Myeloblast;

Normoblast (Magnification: A and B: 400×; C-F: 1000×).

[0023]FIG. 10 are photomicrographs of the same cell preparation as FIG.9 with a different site set up. M: Megakaryocytes;

Basophil erythroblast;

Polychromatophil erythroblast;

Normoblast (Magnification: 1000×).

[0024]FIG. 11 is a graph of optical density (405 nm) of Epo and Epo-bpin serum or plasma samples. Error bars represent standard error (SE).

DETAILED DESCRIPTION OF THE INVENTION

[0025] Despite the availability of recombinant human Epo and full-lengthhuman Epo receptor cDNA clones, little is known about the interaction ofEpo and EpoR, or the signal transducing mechanisms involved inproliferation and differentiation of erythroid progenitor cells.

[0026] Plasmid expression vectors permit expression of a protein fromcloned coding sequences that have been inserted into the vector.Expression vectors generally have a selectable marker and a replicationorigin for selection and maintenance of the vector in a host cell, aswell as inducible regulatory elements for inducing high level expressionof a polypeptide suitable for fusing to an inserted gene. It ispreferred that convenient restriction sites be engineered into thevector downstream from a proteolytic cleavage site sequence. A preferredpolypeptide to be fused to the EpoR coding sequence fragment isglutathione S-transferase, possessing a thrombin proteolytic cleavagesite at the carboxyl terminus.

[0027] An expression vector for the invention disclosed herein expressesthe EpoR extracellular domain as part of a fusion protein that cansubsequently be cleaved to yield purified EpoR extracellular domain. Thecoding sequence for the EpoR extracellular domain may be engineered inany manner suitable for inserting the sequence in the appropriatereading frame in the expression vector. For example, a pair ofpolymerase chain reaction (PCR) primers may be synthesized, such thatthe first primer corresponds to the coding sequence at the 5′ end of theextracellular domain and the second primer is complementary to thecoding sequence of the 3′ end of the extracellular domain. The primerspreferably have convenient restriction enzyme sites flanking theportions of the primers corresponding to the ends of the desired targetsequences. The primers are used to amplify the EpoR extracellular domainfrom a full length human EpoR cDNA template. The resulting PCR productis then cloned into an expression vector. It is preferable to synthesizePCR primers having different restriction sites at each end, rather thanthe same restriction site. The presence of different restriction sitesat each end of the PCR product facilitates the insertion of the humanEpoR coding sequence fragment in the sense orientation.

[0028] High level expression of a fusion protein having humanerythropoietin receptor extracellular domain as part of the fusionprotein is achieved by inducing expression from the recombinant plasmidexpression vector in a host cell culture. A fusion protein ishereinafter referred to as EpoRex-th and a purified human erythropoietinreceptor extracellular domain hereinafter is referred to as Epo-bp(Epo-binding protein). A cell protein extract is preferably preparedfrom an expressing E. coli culture in any suitable manner. EpoRex-th maybe purifed from the extract as desired. For example, the extract may bepassed over a column having the ability to bind the portion of thefusion protein upstream of the Epo-bp coding sequence. The fusionprotein will bind to the column, while other proteins in the extract areeluted in column washes with a buffer that allows binding of fusionprotein to the column matrix. EpoRex-th can be subsequently eluted inhigh purity by changing the buffer conditions.

[0029] Purification of Epo-bp may be accomplished by cleaving purifiedEpoRex-th using an appropriate cleavage method. For example, thecleavage site between the upstream polypeptide and Epo-bp may besensitive to cyanogen bromide or, alternatively, may be sensitive tosite-specific protease cleavage. In a preferred embodiment, a thrombinproteolytic cleavage site is engineered into the upstream polypeptide,but 5′ to the convenient restriction cloning sites positioned at thecarboxyl terminus of the upstream polypeptide coding sequence.

[0030] The cleaved Epo-bp polypeptide segment may be separated from theupstream polypeptide segment by purification techniques such as sizeexclusion chromatography, isoelectric focusing, or affinitychromatography. Furthermore, more than one purification technique may beused, if desired, to achieve the appropriate degree of purification. Apreferred purification technique is affinity chromatography. Forexample, a protease-treated fusion protein mixture may be applied to acolumn having agarose beads coupled to Epo. The cleaved Epo-bp segmentwill bind to the Epo-agarose, while the upstream polypeptide segmentwill pass through the column. Epo-bp may then be eluted by lowering thepH of the liquid phase.

[0031] In an embodiment of the invention, the coding sequence for aminoacids 25 through 250 of human EpoR (hEpoR) is cloned into pGEX-2T(Pharmacia, Mechanicsburg, Pa.). pGEX-2T has an IPTG inducible promoteroperably linked to a coding sequence for glutathione S-transferase(GST). The 3′ end of the GST coding sequence has a thrombin proteolyticcleavage site in the correct reading frame, as well as convenientcloning sites for inserting a coding sequence to be covalently coupledto GST.

[0032] A PCR product having amino acids 25 through 250 of hEpoR is madefrom a suitable DNA template, for example a full-length human EpoR cDNA.A PCR primer is sythesized having the 5′ end of the extracellular domaincoding sequence as well as a BamH1 site, and a PCR primer is synthesizedhaving sequence complementary to the 3′ end of the extracellular domaincoding sequence as well as an EcoR1 site. The BamH1 site in pGEX-2T ispositioned 5′ to the EcoR1 site relative to the GST coding sequence. ThePCR product is cloned into pGEX-2T, and a transformed E. coli colonyhaving a plasmid of the expected size is identified.

[0033] A fusion protein having an amino terminal GST segment and acarboxy terminal EpoR extracellular domain segment is expressed intransformed E. coli by inducing transcription with IPTG. IPTGderepresses the lac promoter positioned upstream of the fusion proteincoding sequence. After allowing expression for a period of timesufficient to accumulate an amount of the fusion protein, cells arelysed and a crude extract is made in any suitable manner. The crudeextract mixture has the fusion protein in addition to many othercellular proteins. The fusion protein, EpoRex-th, may be purified fromthe extract as desired.

[0034] In a preferred embodiment, EpoRex-th is passed over a columnhaving agarose beads coupled to glutathione (GSH). GSH is a substratefor GST, and the GST segment of EpoRex-th will bind to the immobilizedGSH with high affinity. Thus, the fusion protein becomes bound to thecolumn, while virtually all other proteins in the extract will not bind.After washing, EpoRex-th may be eluted from the column by adding reducedGSH to the liquid phase.

[0035] In an embodiment of the invention, purified human erythropoietinreceptor extracellular domain polypeptide may be made by digestingEpoRex-th with thrombin. The resulting digested mixture of GST andEpo-bp may then be applied to an Epo affinity column. The Epo-bp bindsto its ligand, Epo, whereas GST passes through the column. Epo-bp may beeluted in purified form through use of an appropriate elution buffer,for example 0.1M glycine, pH 3.0.

[0036] Antibodies

[0037] Antibodies to human erythropoietin receptor extracellular domaincan be made by presentation of a purified preparation of such apolypeptide to the immune system of an animal. For example, purifiedEpo-bp may be injected subcutaneously, intramuscularly orintraperitoneally into animals such as rats, mice, rabbits, goats, orsheep. Booster injections can be given at intervals, if desired.Circulating antibodies against Epo-bp are made by the immune system ofthe injected animal, and these antibodies can be collected from theblood, and, preferably, from serum. Anti-Epo-bp serum can be used todetect Epo-bp in various assay formats, such as Western blots, ELISAassays and the like. Epo-bp to be detected may be from, for example, apurified preparation of Epo-bp, a bacterial or eukaryotic cell extract,a eukaryotic cell from an in vitro cell culture, a serum sample, or evena tissue or cell biopsy taken from an individual. Anti-Epo-bp antibodiesare expected to recognize the extracellular domain of intact human EpoRas well as Epo-bp. Monoclonal antibodies directed against Epo-bp can bemade by methods known in the art. D'Andrea et al., Blood, 75: 874-80(1990); U.S. Pat. No. 4,558,005; and Harlow and Lane, Antibodies—LabManual, Cold Spring Harbor Laboratory, 1988.

[0038] For example, as described herein, anti-Epo-bp and anti-Epoantibodies were developed in sheep by inoculating Epo-bp or syntheticEpo every three to four weeks for three months in sheep, then collectingserum for antibody purification. The polyclonal antibodies were furtherpurified for the Fab fraction that was fluorescein labeled.

[0039] Antibodies directed against Epo-bp preferably have a specificbinding affinity for the EpoR extracellular domain. For example, serumfrom an animal injected with purified Epo-bp should provide detectablebinding to Epo-bp in Western blots when 10 μg of purified Epo-bp areelectrophoresed in a polyacrylamide gel and exposed to a 1:2000 dilutionof the anti-Epo-bp serum.

[0040] Antibody fragments having specific binding affinity for Epo orEpoR can be generated by known techniques. For example, such fragmentsinclude, but are not limited to, F(ab′)₂ fragments that can be producedby pepsin digestion of the antibody molecule and Fab fragments that canbe generated by reducing the disulfide bridges of F(ab′)2 fragments. Ingeneral, purified immunoglobulins can be added to tubes containingimmobilized papain and incubated at about 37° C. with agitation. Papainis in a phosphate digestion buffer containing cysteine hydrochloride.Solublized Fab fragments can be recovered using a separator tube andpurified by protein A chromatography.

[0041] It should be noted that the sets of antibodies that are used inthe assays (capture and detector antibodies) can be from the same orfrom different preparations of antibodies. For example, both the captureand detector antibodies can be purified from sheep, or the captureantibody can be purified from sheep while the detector antibody ispurified from goat (or vice versa).

[0042] Methods for Measuring Epo and EpoR and Portions Thereof

[0043] Although Epo and EpoR have been cloned and many studies have beencarried out, no methods are available to distinguish between Epo-relatedand EpoR-related clinical problems. Predictions that are made typicallyare based on deduced speculation with clinical symptoms and standardblood tests. Thus, a reliable test method for detecting Epo and EpoR isneeded. The present methods are appropriate for use at the clinical sitefor both rapid and sensitive detection of relevant proteins andantibodies in different disease states. The methods of the invention canbe used to detect the presence, absence, or amount of Epo and EpoR.

[0044] As described herein, a polymerase chain reaction (PCR) basedmethod can be used for detecting Epo or EpoR, including Epo-bp. PCRrefers to a procedure or technique in which target nucleic acids areamplified. Sequence information from the ends of the region of interestor beyond typically is employed to design oligonucleotide primers thatare identical in sequence to opposite strands of the template to beamplified. PCR can be used to amplify specific sequences from DNA aswell as RNA, including sequences from total genomic DNA or totalcellular RNA. Primers are typically 14 to 40 nucleotides in length, butcan range from 10 nucleotides to hundreds of nucleotides in length.General PCR techniques are described, for example in PCR Primer: ALaboratory Manual, Ed. by Dieffenbach, C. and Dveksler, G., Cold SpringHarbor Laboratory Press, 1995.

[0045] In the PCR based method, labeled substrate and antibodies can beused to detect ligand-binding sites and to measure Epo/EpoR mRNAproductions. Primers used in the reaction can be chemically synthesizedusing standard techniques, based on the sequences encoding Epo and EpoR,which are known and described above. In some embodiments, biotinylatedprimers can be used. For example, PCR reactions can be performed understandard conditions using a digoxigenin labeled nucleotide and abiotinylated primer to generate PCR products that are biotinylated andthat contain digoxigenin. The PCR products can be immobilized on asubstrate such as a microtiter plate that is coated with streptavidin oravidin. Immobilization typically is performed by incubating the PCRproduct in the presence of the prepared substrate, for a suitable amountof time e.g., at about 37° C. for 30 minutes. Products are detected byaddition of an anti-digoxigenin antibody or a fragment thereof. Theanti-digoxigenin antibody can be conjugated to an enzyme such ashorseradish peroxidase or alkaline phosphatase to facilitate easydetection of products. In general, a peroxoidase substrate such asdiaminobenzidine or a phosphatase substrate such as4-methylumberlliferyl phosphate (MUP) or disodium p-nitrophenylphosphate (NPP), is added to the reacted sample and color development ismonitored visually, spectrophotometrically at 405 nm when NPP is used asthe substrate, or spectrofluorometrically with a 365 nm excitationfilter and a 450 nm emission filter when MUP is the substrate.Chemiluminescent detection of the PCR products also can be performedwith a chemiluminescent substrate such as Luminol.

[0046] Another method for measuring Epo or EpoR including Epo-bp is animmunofluorescent labeling method. This method includes coating captureanti-Epo-bp fragments and anti-Epo fragments onto a substrate (e.g.,microtiter plate) in different discrete regions. Typically, the antibodyfragments are coated onto a substrate by incubating the antibodyfragments with the substrate for about two hours at room temperature. Abiological sample such as serum/plasma, homogenized tissue, or cells,including blood and bone marrow cells, are added to the coated substrateand incubated for a suitable amount of time. For example, the samplescan be incubated for about 20-30 minutes at temperatures of about 24° C.to about 37° C. Fluorescently labeled detector antibodies (i.e., labeledanti-Epo and anti-EpoR antibodies) then are added, and the materials areincubated for a suitable amount of time (e.g., 10 to 30 minutes at 37°C.). The samples can be washed in an appropriate buffer such as 20 mMphosphate-buffered saline (PBS) with or without albumin. Positivesamples can be identified by observation under a fluorescent microscopeor by determining the absorbance at 405 nm using a microplatefluorometer.

[0047] The immunofluorescent labeling method also can be used to detectanti-Epo antibodies and anti-EpoR antibodies. To detect antibodies, thesubstrate is coated with Epo and EpoR (antigen) in different, discreteregions using the same method as described above. Sample is added, andfluorescently labeled antigen is added. Positive samples are identifiedin the same manner as for detecting Epo and EpoR.

[0048] Suitable fluorescent labels include, for example, fluoroscein,7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™ (MolecularProbes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, lissaminerhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate(FITC), 7-diethylaminocoumarin-3-carboxylic acid,tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, phycoerythrin(B-, R-, or cyanine-), allophycocyanin, Oregon Green™, and Cascade™ blueacetylazide (Molecular Probes, Inc., Eugene, Oreg.).

[0049] An additional method for detecting Epo or EpoR, including Epo-bp,is an enzyme immunoassay. The method includes use of magneticseparation, a technique that is highly efficient and specific for tissuetyping, cell sorting, subcellular organelle fractionation, and DNAseparation. Sensitivity of this method is expected to be 1000-foldenhanced when compared with the other methods available on the market,including the ELISA method. Epo and EpoR or Epo-bp, or anti-Epoantibodies and anti-EpoR antibodies can be detected in this method. Ingeneral, capture beads, e.g., agarose beads, are prepared by coatingwith either Epo or EpoR or a portion thereof for detecting anti-Epo oranti EpoR antibodies, or with anti-Epo or anti-EpoR antibodies fordetecting Epo or EpoR. A biological sample is applied to the capturebeads and incubated at an appropriate temperature and for an appropriatelength of time, e.g., room temperature for about 25 minutes. Then, aspecies-specific, secondary antibody conjugated to an enzyme is added.After incubation and washing, enzyme substrate is added and color isdeveloped. Beads can be pelleted and color is detected as describedabove.

[0050] Numerous variations of these methods can be performed. Forexample, detector antibodies can be biotinylated such that positivesamples can be detected by addition of streptavidin or avidin conjugatedto an enzyme and color development. Horseradish peroxidase conjugatedmolecules also will catalyze the oxidation of Luminol and light can bedetected via chemiluminescence. The emission of light can be enhanced byinclusion of chemical enhancers.

[0051] The purified extracellular domain of EpoR disclosed herein is thefirst such pure human Epo receptor fragment (i.e., free of non-human ornon-Epo receptor amino acid sequence) to be obtained. The experimentsdisclosed herein demonstrate that such a fragment retains the ability tospecifically bind human Epo. The proteins and antibodies disclosedherein are useful for understanding the mechanisms of Epo-Epo receptorinteraction. The purified Epo-bp of the present invention is also usefulfor investigating the structure of the Epo receptor and for identifyingfactors involved in regulating differentiation and proliferationmechanisms in erythroid progenitor cells. Moreover, the inventiondisclosed herein is useful for identifying and quantitating Epo and Eporeceptor, as well as in understanding hematopoietic malignancy andcertain cardiovascular system disorders. In addition, a major discoverydescribed herein is that all human progenitor blood cells have beenshown to contain Epo receptors and are able to binding Epo.

[0052] The invention will be further understood with reference to thefollowing illustrative embodiments, which are purely exemplary, andshould not be taken as limiting the true scope of the present inventionas described in the claims.

EXAMPLES Example 1 Materials and Methods

[0053] Glutathione (GSH)-agarose, pGEX-2T expression vector and SephadexG-50 were purchased from Pharmacia (Mechanicsburg, Pa.). PCR reagentswere from Perkin-Elmer Cetus (Norwalk, Conn.) and Affigel 15 was fromBioRad (Richmond, Calif.). Bacteriophage T4 DNA ligase, restrictionenzymes and isopropylthio-.beta.-D-galactoside (IPTG) were purchasedfrom BRL Gibco (Gaithersburg, Md.). Geneclean II was from Bio 101, LaJolla, Calif. Nitrocellulose was from Schleicher & Schuell Co. (Keene,N.H.). Chemiluminescence (ECL) reagents and .sup.125 I-Epo were fromAmersham (Arlington Heights, Ill.) and unlabeled Epo was a gift ofChugai-Upjohn (Rosemont, Ill.). Phenylmethylsulfonylfluoride (PMSF),diisopropylfluorophosphate (DFP), thrombin, trypsin and Triton X-100,were from Sigma Chemical Company (St. Louis, Mo.). Biotinylated rabbitanti-sheep antibodies and avidin-horseradish peroxidase were from PierceCo. (Rockford, Ill.). LAP37, a full-length human erythropoietin receptor(EpoR) cDNA preparation, was provided by Dr. Bernard G. Forget, YaleUniversity, New Haven, Conn. All other chemicals were of reagent grade.

Example 2 Construction of EpoR cDNA Recombinant Vector

[0054] A recombinant plasmid expression vector, pJYL26, was constructedfrom a PCR product having the human Epo receptor extracellular domaincoding sequence and from the plasmid vector pGEX-2T. The construction ofthis plasmid is explained below.

[0055] PCR amplification was carried out using a full-length human EpoRcDNA, LAP37, as a template. The 5′-sense primer was5′-TTGGATCCGCGCCCCCGCCTAAC-3′ (SEQ ID NO: 1). This primer has a BamH1linker sequence at the 5′ end, followed by the coding sequence for aminoacids 25 through 29 of the full length human EpoR protein. The3′-antisense primer was 5′-TGAATTCGGGGTCCAGGTCGCT-3′ (SEQ ID NO: 2).This primer has an EcoR1 linker followed by sequence complementary tothe coding sequence for amino acids 226 through 222 of full length EpoR.Using a Perkin Elmer-Cetus PCR kit, PCR was carried out with 0.1 μg ofLAP37 cDNA, 20 pM of each primer, 1.25 mM dNTP mixture (dGTP, dCTP, dTTPand dATP), 0.5 μl of Taq polymerase, and 10×buffer supplied in the PCRkit. Amplification was carried out by a PTC-100 Programmable ThermalController, (M. J. Research, Inc. Watertown, Mass.), with denaturationat 94° C. for 1 min, annealing at 55° C. for 1 min and extension at 72°C. for 1½ min, repeated for 25 cycles.

[0056] The sizes of the PCR product (˜600 bp) and pGEX-2T (˜4.9 kb) wereverified on 1% Seakem and 2% Nusieve agarose (FMC Bioproducts, Rockland,Me.) gels running in 1×TA buffer (50×TA in 1 liter volume containing 242g Tris-base and 57.1 ml acetic acid), with a Hae III standard. Both thePCR product and pGEX-2T were purified from gel slices by the GenecleanII method as described by the manufacturer (Bio 101, La Jolla, Calif.).Concentrations of the PCR product and pGEX-2T were estimated byabsorbance readings at OD260. Both DNAs were then digested with BamH1and EcoR1 for 4 hours at 37° C. before ligation. The digested productswere analyzed on 1% Seakem and 2% Nusieve agarose gels. Both the PCRproduct and pGEX-2T fragments were cut from the gel and purified againby the Geneclean II method.

[0057] The ligation was done in a mixture having 1 μg/μl each of PCRproduct and pGEX-2T. The mixture was incubated at 45° C. for 5 minutesand chilled to 0° C. Then, in a 10 μl final volume, 1 μl each of10×bacteriophage T4 DNA buffer and 10×bacteriophage DNA ligase, and 10mM ATP were added. The whole mixture was then incubated at 16° C. in acirculating water bath overnight. Productive ligation was verified byelectrophoresis in a 1% agarose gel in 1×TA buffer running at 100 voltswith lanes containing size standards, pGEX-2T, PCR product, and theligated product (PCR product+pGEX-2T). The ligated product was verifiedto be ˜5.5 kb. An aliquot of ligation mixture was then transformed intoE. coli strain JM109 (20 μg ligation mixture/200 μl JM109). For thetransformation, the E. coli mixture was incubated on ice for 30 minutesafter mixing gently by inverting, and incubated at 42° C. exactly 90seconds. Then the mixture was chilled on ice for 1-2 minutes and 500 μlLB medium (for 1 liter, 10 g bacto-tryptone, 5 g bacto-yeast and 10 gNaCl, pH 7.5, autoclave) was added. After incubating at 37° C. for 45minutes, the LB mixtures were spread on LB/Amp agar petri plates inamounts of 50, 75, 125, 150, and 300 ml of LB mixture. Agar petri plateswere prepared with 20-30 ml of LB/Amp medium, containing 15 g agar/literLB (autoclaved) and 100 μg/liter ampicillin. Control LB/Amp plates weremade with intact pGEX-2T, digested pGEX-2T and PCR product only. Theplates were kept on the bench top to absorb liquid for a few hours andinverted plates were incubated at 37° C. for 24 hours. Grown colonieswere seeded on gridded plates, which were incubated again at 37° C. for24 hours, while another set of all colonies was grown in 5 ml each ofthe LB/Amp medium overnight.

[0058] The DNA was extracted from each colony by the miniprep method.Each colony was cultured overnight with 5 ml LB/Amp medium (2 μl/ml of50 μg/ml Amp stock) in a loosely capped 15-ml plastic tube in avigorously shaking 37° C. incubator. The following day, 1.5 ml of eachculture was pelleted in a microfuge for 3 minutes at 4° C. at 14,000×g,and resuspended in 93 μl STET plus 17 μl of lysozyme stock (STET: 5%sucrose+5% Triton X-100+50 mM Tris, pH 8.0+50 mM EDTA, pH 8.0, stored at4° C.; lysozyme stock: 5 mg/ml, stored in a freezer). The resuspendedmixture was then incubated for 10 minutes at room temperature and boiledfor 2 minutes before spinning in a microfuge at 4° C. for 15 minutes at14,000×g. The pellet was removed with a sterile tooth pick, 2 μl ofRNAse (100 mg/ml) was added to the supernatant, followed by incubationat 37° C. for 30 minutes. After incubation, 110 μl of ice-coldisopropanol was added and the mixture was inverted 4 times beforepelleting at 14,000×g, 4° C. for 15 minute. The pellet (DNA) was thenwashed with ˜1 ml of 70% ethanol to remove residual STET and othercontaminants, and the pellet centrifuged again at 14,000×g, 4° C. for 15minutes. The pellet was then air dried for 1-2 hours and resuspended in25 μl of sterile dH₂O.

[0059] The extracted DNAs were verified on a 0.8% agarose gel in TAbuffer, running at 100 volts until the front dye line migrated ⅘ of thelength of the gel. The gel was stained with ethidium bromide (0.5 μg/ml)at room temperature for 15 minutes on a gentle shaker and destained withdH₂O for 15 minutes. DNA bands were examined under UV light. Cultureshaving DNA of the expected size were examined in 1% agarose gels runningin TA buffer after EcoR1 and/or EcoR1 plus BamH1 digestion. The EcoR1and BamH1 digestion was done by incubating the sample mixture at 37° C.water bath for 2 hours with the mixture of 1 μg of EcoR1 or BamH1 per 2μg of DNA in 1 μl/10 μl sample volume of 10×reaction buffer provided inthe restriction enzyme kit. One colony having a plasmid of about 5.5 kbin size was selected after examining both EcoR1 and EcoR1 plus BamH1digested DNA sizes in 1% agarose gels. The plasmid in this colony wasnamed pJYL26. A diagram of pJYL26 is shown in the upper part of FIG. 1.

Example 3 Purification of EpoRex-th Fusion Protein

[0060] This example teaches the production and purification of a fusionprotein having two segments. The first segment is a polypeptide, GST,with a thrombin cleavage site at the carboxyl terminus. The secondsegment, fused to the first segment at the thrombin cleavage site, isthe extracellular domain of human Epo receptor. The fusion proteinEpoRex-th, containing GST and Epo-bp, is purified by GSH-agaroseaffinity chromatography.

[0061] Transformed E. coli containing the recombinant vector pJYL26 weregrown overnight at 37° C. with vigorous shaking in 400 ml of LB mediumwith 100 μg/ml of ampicillin. The following day, the culture was dilutedin 4 liters of fresh LB/Amp media and incubated for another 90 minbefore adding 1 mM isopropylthio-β-D-galactoside (IPTG). After 4 hoursof IPTG induction, the cells were pelleted at 3,000×g at 4° C. for 15min and resuspended in 160 ml of lysis buffer, containing 50 mM sodiumphosphate, pH 7.4, 10 mM β-mercaptoethanol (βME), 10 mM EDTA, pH 8.0, 1mM PMSF and 1 mM DFP. 160 mg of solid lysozyme was then added. Using a60 cc syringe, the lysed cell suspension was homogenized by passingthrough 18, 21 and 23 gauge needles three times, and incubated on ice 30min. After dry ice/methanol freeze thaw at 37° C. for 3 times and mildsonication, 1% of Triton X-100 was added. The supernatant was collectedby centrifugation 15×kg at 4° C. for 15 min.

[0062] A GSH-agarose column was prepared by washing swollen GSH-agarosebeads 3 times with 10 bed volumes of phosphate-buffered saline (PBS: 16mM Na₂HPO₄, 4 mM NaH₂PO₄, pH 7.4 in excess salt of 3M NaCl) to removepreservatives and elutable dextran from the agarose. The column was thenequilibrated with 5 bed volumes of isotonic PBS. The IPTG inducedextract was applied to the column and the column was washed twice with 5bed volumes of PBS, which elutes all proteins with no affinity forGSH-agarose EpoRex-th was then eluted by adding 5 bed volumes of elutionbuffer, containing 5 mM reduced GSH in 50 mM Tris-HCl, pH 8.0. Fractionsof 1.0 ml were collected and the A₂₈₀ was determined for each fraction.FIG. 2a shows the A₂₈₀ data. Fractions 18-23 were subsequently shown tohave the EpoRex-th protein. These fractions were pooled. From afour-liter cell culture preparation, an average of 2 mg of EpoRex-th wasextracted.

Example 4 Purification of Epo-bp

[0063] EpoRex-th contains a thrombin-specific proteolytic cleavage site,as diagrammed in the lower half of FIG. 1. Thrombin cleaves specificallyat the sequence -CTG GTT CCG CGT GGA TCC- (SEQ ID NO: 3), which codesfor the amino acids Leu Val Pro Arg Gly Ser, as shown in FIG. 1. Smithand Johnson, Gene 67: 31-40 (1988). Thrombin was incubated withEpoRex-th to cleave the GST segment from the Epo-bp segment and the twosegments were purified by Epo-agarose affinity, as described below.

[0064] Various thrombin concentrations were tested in order to find themost effective range of thrombin cleavage. Purified EpoRex-th wasincubated with 0.0125, 0.125, 0.6 or 2.4 μg of thrombin per 60 kgEpoRex-th at room temperature or 37° C. for 1 hour in PBS buffer, pH7.4. The results were analyzed by polyacrylamide gel (12.5%)electrophoresis. After staining with Coomassie blue, bands could be seencorresponding to the fusion protein EpoRex-th (55 kDa), Epo-bp (29 kDa)and GST (26 kDa). The 0.6 μg concentration was selected for completedigestion of EpoRex-th. The results are presented in FIG. 3.

[0065] For thrombin cleavage, 60 kg of EpoRex-th was incubated at roomtemperature for 1 hr with 0.6 μg thrombin. The mixture was applied to anerythropoietin-agarose column in Tris buffered saline (TBS) or PBS.Epo-bp was eluted with 0.1M glycine buffer, pH 3.0. Fractions of 0.5 mlwere collected into tubes, containing 0.5 ml of 2M Tris-HCl, pH 7.5.Epo-bp peak fractions 14-19 were pooled and then dialyzed overnight inTBS or PBS at 4° C. for further experiments. Approximately 200 μg Epo-bpwas extracted, starting from a four-liter cell culture preparation.

[0066] The Epo-agarose column was prepared from Epo-agarose beads. TheEpo-agarose beads were prepared by overnight dialysis of Epo (0.5 mg/ml)in 0.1M 3 (N-morpholino)-propanesulfonic acid (MOPS) at 4° C. Epo waslinked to Affigel 15 beads by admixing 1 ml of the dialyzed Epo-solutionand 2 ml of washed Affigel 15, and incubated at room temperature for 2hours on a rotating shaker. The supernatant was removed aftermicrocentrifuging at 2000×g for 30 sec. The packed Epo-agarose beadswere washed 3 times in TBS or PBS at 4° C. and stored until ready touse. After collecting desired protein fractions, Epo-agarose beads maybe washed extensively with TBS or PBS and stored at 4° C. or reuse.

Example 5 Production of Antibodies to Epo-bp

[0067] This example teaches the production of antibodies directedagainst purified Epo-bp. Purified Epo-bp is electrophoresed in a 12.5%SDS-PAGE gel and the Epo-bp protein band is resuspended in PBS andinjected into sheep. Sheep serum having anti-Epo-bp antibody is shown todetect purified human Epo-bp when the serum is diluted 1:2000.

[0068] Epo-bp (0.5 mg), purified as described above, was mixed with2×treatment (Laemmli) buffer and boiled for 10 minutes. The mixture wasapplied to a 12.5% SDS gel and electrophoresed at 200 volts for 3-4hours. The gel was stained with 0.125% Coomassie blue overnight,destained 1-2 hours with dH₂O, and the Epo-bp band cut out of the gelwith a razor blade.

[0069] The Epo-bp gel slice was resuspended in 10-15 ml of PBS bufferand passed through a syringe repeatedly until the gel was crushed intosmall pieces forming a suspension mixture with PBS. The suspension wasinjected subcutaneously in adult sheep. Epo-bp was injected at a ratioof 0.5 mg Epo-bp or more per 25 kg weight of the animal. Two boosterinjections, with the same dose as in the initial injection, were givenonce every 3 weeks following initial injection. After the second boosterinjection, blood can be withdrawn for collection of antibodies.Injections can be given every month to maintain antibody production bythe animal. Injection sites are rotated on the animal. Sambrook et al.,Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press, Chapter18, 1989.

[0070] To obtain blood from injected animals, hair at the blood samplingsite was cleaned with 70% alcohol. Ear arteries or other accessiblearteries were shaved. Sheep can be bled via the jugular vein usinggravity withdrawal in a vacuumed bottle, and serum is separated. A smallamount of xylene was applied to the tip of the ear but not at thebleeding site. Blood was gently withdrawn with a butterfly and put intoa glass tube having no heparin. The blood was incubated at roomtemperature for 1 hour to allow clotting, the clot was loosened from thetube wall with a pasteur pipet, and the tube was incubated at 4° C.overnight. The clotted blood mixture was poured into a dish and the clotremoved. The unclotted remainder was returned to the glass tube andcentrifuged at 3000 rpm for 10 minutes. The supernatant (serum) wasapplied to an Epo-bp-affinity column and antibodies binding to thecolumn were eluted by with 0.1M glycine buffer, pH 3.0, using the sameprocedures as discussed above for purification of Epo-bp. The eluate wasdialyzed in PBS overnight at 4° C. and stored at −70° C. in 500 μlaliquots. The Epo-bp affinity column was prepared from Epo-bp andAffigel 15 agarose beads in the same manner as the Epo-bp Affigel beadsdescribed in Example 6 below.

[0071] Solutions used in this example are prepared as follows: LysisBuffer II: 50 mM NaPO₄ (7.74 ml of 0.5M dibasic PO₄ plus 2.26 of 0.5Mmonobasic PO₄)+10 mM β-mercaptoethanol+10 mM EDTA, pH 8; PBS Buffer:0.15M NaCl+16 mM dibasic PO₄+6 mM monobasic PO₄, pH 7.4; TBS buffer: for1 liter, 12.5 ml of 2M Tris-HCl, pH 7.4+27.5 ml of 5M NaCl; 2×Treatment(Laemmli) buffer: 0.125M Tris-HCl, pH 6.8+4% SDS+20% glycerol+10%β-mercaptoethanol.

[0072] Sheep anti-Epo-bp serum was analyzed for binding to purifiedEpo-bp by Western blotting as described in Sambrook et al., MolecularCloning, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989 and inWestern blotting protocols provided by the ECL manufacturer, AmershamCo., Arlington Heights, Ill. Following thrombin cleavage, EpoRex-th andEpo-bp were separated electrophoretically on an SDS-PAGE gel. The gelwas then blotted onto nitrocellulose (Schleicher and Schuell Co., Keene,N.H.). Sheep anti-Epo-bp serum was added to the nitrocellulose in Blotto(for 1 liter: 80 g non-fat dry milk, 30 ml 5M NaCl, 10 ml 2M Tris-HCl,pH 7.5 and 0.05% Tween-20) at a 1:2000 dilution and incubated at roomtemperature for 1 hour with gentle agitation. After rinsing off thefirst antibody, a second reagent, biotinylated rabbitanti-immunoglobulin anti-sheep (1:10,000 dilution) antibody was added tothe nitrocellulose in Blotto, and incubated at room temperature foranother 1 hour with rocking. Horseradish peroxidase-avidin (1:10,000dilution) was added and the mixture incubated at room temperature for 45min. After soaking the washed nitrocellulose briefly inchemiluminescence (ECL) reagents, wet blots were exposed immediately onKODAK X-ray film. FIG. 4 shows a photograph of the Western blot, withthe lanes having the following proteins applied: Lane 1, molecularweight standards; Lane 2, thrombin digested EpoRex-th; Lane 3, GST; Lane4, purified Epo-bp. As shown in lane 4 of FIG. 4, purified Epo-bp wasdetected by a 1:2000 dilution of anti-Epo-bp antibody. The apparentmolecular weight of the purified Epo-bp was about 29 kDa.

Example 6 Binding of Epo to Epo-bp

[0073] Ligand binding of Epo to Epo-bp and effects of Epo concentrationon binding are taught in this example.

[0074] Epo-bp beads were prepared by adding 60 μg/ml Epo-bp to washedAffigel 15 agarose beads in PBS, with a final concentration ofapproximately 30 μg of protein per 1 ml of Epo-bp beads. The mixture wasincubated at room temperature for 2 hours on a rotating platform. Afterwashing 3 times with ice cold PBS buffer, the pellet was resuspended in1 ml of PBS buffer. For binding assays, 30 μl of the final suspension(approximately 1.0 μg of Epo-bp) were admixed with variousconcentrations of ¹²⁵I-Epo and incubated for 1 hour at room temperaturewhile resuspending every 5 min with a pipet. At the end of theincubation, 1 ml of ice cold PBS buffer was added to wash out unreacted¹²⁵I-Epo and the wash was repeated twice more. The reacted beads werecounted by a gamma counter. Proteins smaller than the intact Epo-bp fromtrypsin digested extracts (see below) were also applied in the same wayto test any effect on ligand binding. Nonspecific binding was measuredby the same method except the mixture was preincubated with a 200-foldexcess of unlabeled Epo for 1 hour prior to adding labeled Epo.

[0075] Binding of Epo-bp to Epo is shown in FIG. 5. Each point in FIG. 5is the mean of 2-4 samples. Data are expressed as mean±SEM. A p value ofless than 0.05 was considered significant. Results were analysed withthe two-tailed Student t-test. The specific binding activity of Epo toEpo-bp dramatically increased as Epo concentration increased; thebinding tripled from 8 nM to 12 nM ¹²⁵I-Epo. Apparent saturation of Epobinding occurred at 12 nM. This was also confirmed in the unreactedsupernatant of ¹²⁵I-Epo. Binding of ¹²⁵I-Epo to Epo-bp was significantlyinhibited in the presence of unlabeled Epo at concentrations of 8 nM andhigher of ¹²⁵I-Epo (p<0.0001 in both comparisons). Nonspecific bindingwas somewhat higher than expected. It had been expected that the excessunlabeled Epo might eliminate ¹²⁵I-Epo binding completely because of thesensitivity and specificity of Epo binding to Epo-bp shown in Westernblots and binding assays.

[0076] Trypsin digestion experiments were performed to find a minimumsequence of Epo-bp involved in ligand binding. There are severalarginine and lysine sites in the Epo receptor protein, which may bespecific sites for trypsin digestion. Trypsin digestion of Epo-bp wascarried out at 10, 20, 30, 50, 100 μg and 2 mg of trypsin per 5 μg ofEpo-bp in a total volume of 200 μg in PBS, pH 6.7 at 37° C. for 3 or 6hours. The reaction was stopped by adding the same volume of 2N aceticacid or by boiling. As shown in FIG. 6, Epo-bp was cleaved effectivelywhen 20 μg or more of trypsin was present. Trypsin is visible as a 23.2kDa protein band in the lane having 2 mg of trypsin. The trypsindigested Epo-bp is visible as a 20-kDa protein. In FIG. 6, Lane 1contains standard molecular weight markers; lane 2 is a control; lanes3-8 represent digestions at concentrations of 10, 20, 30, 50, 100 μg and2 mg trypsin, respectively at 37° C. for 3 hours; lanes 9-14 representthe same concentrations of trypsin incubated at 37° C. for 6 hours.

[0077] Since uncut Epo-bp is aproximately 30 kDa, gel filtrationchromatography using Pharmacia Sephadex G-50 (MW 30,000) was applied toseparate protein components of size.ltoreq.30,000 molecular weight fromthe total mixture. A powdered form of Sephadex G-50 was hydrated andwashed several times with isotonic PBS to wash out preservatives.Trypsin digested EpoRex-th was applied to the top of the gel column in atotal volume of 0.2 ml in PBS. The column was centrifuged at 2,000×g for4 min at room temperature in a swinging-bucket rotor. The first effluentwas collected from the bottom of the syringe (about 0.2 ml) into adecapped microfuge tube. This effluent contains proteins having a sizelarger than Epo-bp. Another 0.2 ml of PBS buffer was added to the columnand a second eluate collected into a new decapped microfuge byrecentrifuging for 10 min. This step was repeated twice. The secondeluate was applied to an Epo-agarose column and peak fractions wereexamined by SDS-PAGE gels and Western blotting. The final product ofEpo-bp, as a result of trypsin digestion, was approximately 20 kDa,shown in FIG. 6. The antibody did not recognize the cleaved Epo-bp.Thus, deletion of 30 amino acids from Epo-bp by trypsin digestioncompletely eliminated recognition by antibodies to Epo-bp, as verifiedby Western blotting.

Example 7 Detection of EpoR in Progenitor Cells

[0078] PCR reagents were purchased from Perkin-Elmer Cetus Norwalk,Conn.) and Affigel® 15 from BioRad (Hercules, Calif.).Isopropylthio-D-galactoside (IPTG) was purchased from BRL Gibco(Rockville, Md.). Nitrocellulose was purchased from Schleicher & Schuellco. (Keene, N.H.). Chemiluminescence (ECL) reagents were purchased fromAmersham (Arlington Heights, Ill.). 2,7-Dichlorofluoresein,Phenylmethylsulfonyl-fluoride (PMSF), diisopropylfluorophosphate (*DFP),thrombin, Triton X-100, biotin-amidocaproyl hydroazide, alkalinephosphatase conjugate, and disodium p-nitrophenyl phosphate werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Biotinylated rabbitanti-sheep antibodies, avidin-horseradish peroxidase, and IgGpurification kits were purchased from Pierce Chemical Co. (Rockford,Ill.). Digoxigenin (for labeled nucleic acids), streptavidin and rabbitanti-digoxigenin Fab/alkaline phosphate conjugates were purchased fromBoehringer Manheim Corp. (Indianapolis, Ind.), and microplates werepurchased from Corning Costar (Cambridge, Mass.). Sheep anti-Epo andanti-Epo-bp antibodies were prepared as described above in Example 5.Oligonucleotides were synthesized by the microchemical facility of theInstitute of Human Genetics, University of Minnesota, Minneapolis, Minn.All other chemicals were of reagent grade.

[0079] Animals: Five week old male Sprague-Dawley rats were assignedinto control or Epo treatment groups with a light (L) cycle from 04:00to 18:00 and with each subgroup of five rats at 0, 4, 8, 12, 16, and 20hours after L-on. Rats were on a standard rat chow and had freelyaccessible drinking water. After four weeks, body weight was measuredevery four hours just before and immediately after the completion of afour-week course of three times-weekly Epo (50-U/kg BW) or physiologicalsaline injections. For blood pressure measurement, the rat's femoralartery was canulated.

[0080] Statistical Method: Results were analyzed by the two-tailedStudent t-test, and linear least squares rhythmometry. Data areexpressed as mean±SEM. A p value of less than 0.05 was consideredsignificant.

[0081] Results: Every rat treated with 50 units/kg Epo, three timesweekly for four weeks, exhibited splenomegaly (120 rats), while none ofthe saline treated rats exhibited splenomegaly. It should be noted thatthe concentration used in the current study fell within the dosecommonly used in clinical settings (50-150 units/kg). FIGS. 7A-7C arerepresentative of Epo treated spleens, while FIG. 7D is a controlspleen. Blood pressures were elevated overall with Epo treatments.Baseline values of control vs. Epo treatment were 89 vs. 86 mm Hg,whereas post treatment values were 116 vs. 135 mm Hg (p<0.0001). At thegiven values of blood pressure elevation, increased hematocrit, andspleen enlargement, the changes were worse in the early morning (8:00a.m. values, the worst for all the three). Table 1 provides a summary ofthe observations.

[0082] The receptor was labeled by the following method. Bone marrowcells were washed three times in PBS and dispensed at 1-3×10³ cells perwell of round-bottomed microplates for control and samples. Supernatantswere removed and 100 μl of purified Fab-fractionated fluoresceinconjugated anti-Epo-bp antibodies were added. The mixture was incubatedon ice for 30 minutes, and then cells were washed three times by adding400 μl of buffer containing 1% FCS and 0.01% NaN₃ in PBS to each sampleand centrifuged at 200 g for two minutes. The cells were resuspendedonto a vortex in total volume up to 50 μl of PBS and analyzed under aninverted fluorescence microscope. FIGS. 8A-8F are photomicrographs ofcontrol bone marrow cells with or without fluoroscein labeling. FIGS.9A-9F are photomicrographs of bone marrow progenitor cells with orwithout fluorescein labeling. The binding site of the receptor isvisualized using the fluorescent labeling technique. It should benoticed that the binding sites are located among the megakaryocytes (M),erythroblasts

[0083] (

,

), and normoblasts (

) as well as myeloblasts (

). FIG. 10 contains the same preparation as FIG. 9, except that adifferent field is shown. This is the first publication showing the cellreceptor site among the cells and identifying the cell type where thereceptor is located. TABLE 1 Circadian variations of body weight, bloodpressure and hematocrit with Epo treatment EPO Variable Time Rats (n)Control treatment p-value Body weight (g) 0000 5  313 ± 11.6  305 ± 13.0ns 0400 5  305 ± 8.9  294 ± 6.7 ns 0800 5  324 ± 18.4  294 ± 4.9 ns 12005  308 ± 13.0  290 ± 9.0 ns 1600 5  310 ± 10.2  295 ± 13.9 ns 2000 5 317 ± 13.3  291 ± 14.3 ns BP(mm Hg) 0000 5  116 ± 5.8  131 ± 7.6 ns0400 5  120 ± 4.6  131 ± 4.8 ns 0800 5  116 ± 5.8  139 ± 3.9 0.003 12005  108 ± 1.0  128 ± 8.1 0.041 1600 5  119 ± 3.2  140 ± 6.3 0.016 2000 5 118 ± 4.1  137 ± 6.2 0.030 Hematocrit (%) 0000 5   42 ± 2.6   60 ± 4.50.009 0400 5   41 ± 2.3   64 ± 2.2 <0.0001 0800 5   42 ± 1.6   66 ± 2.7<0.0001 1200 5   44 ± 0.5   65 ± 1.5 <0.0001 1600 5   45 ± 1.4   61 ±3.8 0.003 2000 5   43 ± 3.0   64 ± 1.5 0.001 Spleen Weight (g) 0000 50.83 ± 0.2 1.63 ± 0.5 0.01 0400 5 0.82 ± 0.2 1.47 ± 0.7 ns 0800 5 0.88 ±0.1 1.69 ± 0.2 <0.0001 1200 5 0.96 ± 0.2 1.63 ± 0.3 0.004 1600 5 0.73 ±0.1 1.37 ± 0.4 0.004 2000 5 0.92 ± 0.2 1.60 ± 0.6 0.03

Example 8 Immunofluorescent Labeling Method for Detecting Epo or EpoR orPortions Thereof

[0084] Fluorescent labeling of Epo/Epo-bp and their antibodies wascarried out according to manufacturer's instructions. The Fab antibodyfraction that was fluorescein-labeled also was purified as described inthe manufacturer's protocol. The Fc fraction also can be purified andsaved for other uses. These materials were used to detect Epo and EpoRin blood and/or tissue samples, including plasma, serum, megakaryocytes,RBC and WBC progenitors, or homogenized spleen or kidney samples. PBScontaining 16 mM Na₂HPO₄, 4 mM NaH₂PO₄ and 2.7 mM KCl in 0.9% NaCl, pH7.4 was used unless otherwise indicated. Assay results can be ready inless than one hour.

[0085] A. Serum/Plasma/Homogenized Tissue Samples

[0086] Precoat a 96-well microplate with 1 μg/well of purified anti-Epoantibodies for Epo or anti-Epo-bp antibodies for Epo-bp or EpoRdetection. Anti-epo and anti-Epo-bp antibodies are precoated on the96-well microplate in different, discrete regions to facilitatesimultaneous detection of Epo and EpoR.

[0087] 1) Add 200 μl of 1:10 diluted sample (serum/plasma/homogenizedtissue) to capture Epo/antibodies or Epo-bp/antibodies. The mixture isincubated at 37° C. for 20 minutes then supernatant is removed byinverting the plate with a single flicking motion over a sink.

[0088] 2) Add 100 μl/well of 1 μg/ml Fab-fractionated fluoresceinlabeled anti-Epo antibodies for Epo, or Fab-fractionated fluoresceinlabeled anti-Epo-bp antibodies for Epo-bp or EpoR detection, andfluorescein labeled Epo or Epo-bp for anti-Epo or anti-Epo-bp antibodydetection, respectively. Incubate the mixture at 37° C. for 15 minutesand wash three times with 400 μl/well of buffer containing 1% FCS and0.01% NaN₃ in PBS.

[0089] 3) Add 50 μl of PBS buffer and analyze under an invertedfluorescence microscope and/or measure the absorbance at 490 nm using amicroplate fluorometer.

[0090] B. Progenitor Cell Samples

[0091] 1) Washed blood or bone marrow cells in PBS are dispensed at1-3×10³ cells per well of a round-bottomed microplate for control andsample plates, and centrifuged into a pellet at 200 g for two minutesusing a microtiter plate rotor attached centrifuge.

[0092] a. Remove supernatants and add 100 μl of Fab-fractionedfluorescein conjugated antibodies, and mix plate well onto a vortex.Incubate the mixture on ice for 30 minutes, and then wash cells threetimes by adding 400 μl of buffer containing 1% FCS and 0.01% NaN₃ in PBSto each well and centrifuged at 200 g for two minutes.

[0093] b. Resuspend the cells in the plate onto a vortex in total volumeup to 50 μl of PBS and analyze under an inverted fluorescence microscopeand/or measure the absorbance at 490 nm using a microplate fluorometer.

[0094] Fluorescein labeled cells can be analyzed immediately or kept onice for up to two to three hours. FIGS. 9 and 10 show the fluoresceinlabeled receptor sites of bone marrow progenitor cells where thereceptor is visualized.

Example 9 PCR Method of Detection

[0095] Colorimetric substrate and antibodies will be used to detectligand-binding sites and to measure Epo/Epo-R mRNA productions. Forrapid labeling to develop an enzyme immunoassay, nonradioactive labelingof PCR generated probes with Epo/Epo-R DNA can be applied inhybridization to reduce nonspecific background reactions reflectingcross-hybridization between vector sequences. For ELISA-type detectionof DIG modification with digoxigenin-specific alkaline phosphatase (AP)conjugates, the PCR product can be used as nonradioactive hybridizationprobe and, unlabeled target nucleic acid is hybridized to makeDIG-labeled hybridization complex. Antigen-antibody-AP complexes arelocated using the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP)in combination with nitro blue tetrazolium chlorida (NBT), which detectsthe precipitated inoxyl group.

[0096] mRNA Extraction: Total RNA was extracted using total RNA kitsfrom Qiagen Inc. (Chatsworth, Calif.), with equal weight of each organor equal cell numbers (use 2 ml of blood sample to lyse or 1 gram oftissue to homogenize for RNA extraction, according to the manufacturer'sdescription). Samples were prepared to adjust binding conditions andapplied to RNeasy Kit, and washed three times with PBS to elute totalRNA. mRNA will be purified using a Perkin-Elmer Cetus PCR kit with Epoor Epo-R primers and the total RNA from the above extracted sample. ThemRNAs were verified on an agarose gel stained with ethidium bromide. ThemRNA bands are read/quantitated and documented with photographs.

[0097] Stock Solutions:

[0098] BCIP: 50 mg/ml 10% dimethlyformide;

[0099] NBT: 50 mg/ml, 70% dimethylformamide, stored in the dark, 4° C.;

[0100] Alkaline phosphatase (AP): nitroblue tetrazolium chloride (20 μlat 50 mg/ml in 50% dimethylformamide) and 5-bromo-4-chloro-3-indoylphosphate p-toluidine salt (100 μl at 50 mg/ml 50% dimethylformamide)are dissolved in 30 ml of 100 mM Tris-Cl, pH 9.5, 100 mM NaCl, 5 mMMgCl₂ stored at room temperature.

[0101] BCIP/NBT preparation: just before use, add 33 μl of NBT stocksolution to 5 ml of ALP buffer and mix well. Then, add 16.5 μl of BCIPstock solution and mix well. The substrate mixture should be used within30 minutes.

[0102] For optical detection, the optical substrates BCIP/BT are added,resulting in blue color products and measured the absorbance at 405 nm.

[0103] Procedure:

[0104] 1) A PCR kit is provided that includes control template, a pairof Epo and Epo-R PCR primers, Taq DNA polymerase, andDigoxigenin-labeled-dUTP/dNTP. Sample DNA is needed when the test isperformed. Taq DNA polymerase also can be purchased separately fromelsewhere.

[0105] 2) Twenty-five cycles are PCR are performed to produce Dig-DNA,biotinylated DNA, or Dig-biotinylated DNA fragments in a total volume of100 μl. PCR products are verified on, for example, by electrophoresisthrough a 1% agarose gel and ethidium bromide staining.

[0106] 3) A 96-well EIA microplate is precoated with 100 μl ofstreptavidin per well (10 μl/ml in TBS, containing 20 mM Tris-HCl, 150mM NaCl, pH 8.0) for biotinylated DNA detection in the avidin-biotincomplex method or purified Epo/EpoR antibodies for either biotinylatedor Dig-labeled detection.

[0107] 4) Volume of PCR products is increased to 200 μl in TBS.Approximately 100 μl of the PCR product is added in duplicate to the96-well EIA microplates to immobilize. Incubate at 37° C. for 30 minutesto bind PCR products, then supernatants are removed by inverting theplate with a single flicking motion over the sink.

[0108] 5) Add 100 μl/well of alkaline phosphatase-conjugated rabbitpolyclonal sheep anti-digoxigenin Fab (1 μg/ml) and incubate at 37° C.for 30 minutes, then wash three times in 400 μl of TBS.

[0109] 6) Add 100 μl substrate solution. Phosphatase substrate, disodiump-nitrophenyl phosphate (1 mg/ml) in 10% vol/vol diethanolamine-HClbuffer, pH 9.5, will be added for color development. Incubate at roomtemperature for 30 minutes for enzyme reaction and then add 100 μl ofstop solution containing 0.01% NaN₃ and wash three times with 400 μl ofTBS (do not shake while color is developing).

[0110]7) Record color development visually and determine the absorbanceat 405 nm by an ELISA reader.

[0111] Digoxigenin-incorporated DNA fragments are recognized byanti-digoxigenin antibodies.

Example 10 Enzyme Immunoassay (EIA) Method

[0112] Bead Method

[0113] Epo and Epo-bp agarose beads are prepared with overnight dialyzedEpo and Epo-bp (0.5 mg/ml) in 0.1 M 3(n-morpholino)-propanesulfonic acid(MOPS) at 4° C., and linked to Affigel® 15 beads by admixing 1 ml of thedialyzed Epo or Epo-bp solution and 2 ml of washed Affigel® 15. Themixture was incubated at room temperature for two hours on a rotatingshaker. Supernatant was removed after microcentrifuged at 2000×g for 30seconds, and washed three times in PBS or TBS, depending on the nextstep.

[0114] 1) Affigel® 15 beads are coated with 2 μg/well anti-Epo for Epoand vice versa, or anti-Epo-bp antibodies for Epo-bp detection, and viceversa.

[0115] 2) 200 μl of 1:10 diluted sample serum/plasma/homogenized tissuein TBS is added to capture Epo/antibodies or Epo-bp/antibodies,respectively, then incubated at room temperature for 25 minutes with agentle rocking and washed three times with TBS at 200 g for two minutesat 4° C. to remove supernatant.

[0116] 3) 100 μl of 1 μg/ml rabbit anti-sheep immunoglobulins/alkalinephosphatase conjugate is added as an indicator and incubated at roomtemperature for 25 minutes with gentle rocking and washed three timeswith TBS.

[0117] 4) 100 μl of phosphatase substrate, disodiump-nitrophenylphosphate (1 mg/ml) in 10% vol/vol diethanolamine-HClbuffer, pH 9.5, are added for the color development, incubated for 30minutes at room temperature, and the absorbance at 405 nm measured.

Example 11 Avidin-Biotin Complex Method

[0118] Epo, Epo-bp and their antibodies were biotinylated withbiotin-amidocaproyl hydroazide according to the manufacturer'sinstructions. 2.5 mg of each product from chromatography was dialyzedovernight in PBS buffer before the biotinylation. Each dialyzed productwas biotinylated by adding 40 mg biotin-amydocaproyl hydrazide and 5 mgNaCNBH₃ (Sigma, St. Louis, Mo.), and incubated at 37° C. for two hours.Residual reagents were separated from the products by extensive dialysisin a PBS buffer. Detection includes Epo for anti-Epo antibodies,anti-Epo antibodies for Epo, Epo-bp for anti-Epo-bp antibodies andanti-Epo antibodies for Epo-bp detection in blood and/or tissue samples,including plasma or serum progenitors, and homogenized spleen or kidneysamples.

[0119] 1) A 96-well microplate is precoated with 100 μl of 20 μg/ml inPBS per well of purified anti-Epo for Epo or vice versa; or anti-Epo-bpantibodies for Epo-bp detection or vice versa.

[0120] 2) 200 μl of 1:10 diluted sample serum/plasma/homogenized tissueare added and incubated at 37° C. for 30 minutes and then supernatant isremoved.

[0121] 3) 100 μl of 10 μg/ml in PBS per well of biotinylated anti-Epofor Epo or vice versa or anti-Epo-bp antibodies for Epo-bp detection orvice versa. The mixture is incubated at 37° C. for 20 minutes, thenwashed three times with 400 μl PBS.

[0122] 4) 100 μl of streptavidin (2 μg/ml) per well in PBS buffer isadded and incubated at 37° C. for 20 minutes, then washed three timeswith PBS containing 1% FCS and 0.01% NaN₃. The supernatant is removed byinverting the plate with a single flicking motion over a sink.

[0123] 5) The mixtures in the plate are resuspended by vortexing in atotal volume up to 100 μl of the buffer and the absorbance is measuredat 405 nm using a BioRad microplate reader or any equivalent microplatereader. Assay results can be ready in less than one hour.

[0124] Primary antibodies locate target proteins, which are detected byusing species-specific secondary antibodies to conjugate withhorseradish peroxides (HRP). HRP-conjugated streptavidin complexes canalso be used without changes to routine protocols. HRP-conjugatedmolecules (e.g., antibodies, sreptavidin or protein A) will catalize theoxidation of Luminol and detect light emission via chemiluminescencethat can be enhanced by the sustained emission of light provided by theinclusion of chemical enhancers in the HRP-catalized oxidation ofLuminol. ECL gives approximately 1000-fold more light than the oxidationof Luminol alone.

Example 12 EIA for Detection of Epo and EpoR

[0125] Blood samples were obtained from volunteers at the University ofMinnesota laboratories, Twin Cities hospitals and blood bank, as well asfrom Japanese Hospitals in Yokohama and Tokyo. All antibodies and Epo-bpwere prepared as described above. The EIA was performed as follows.

[0126] 1) EIA microplates were coated with 2 μg/well anti-Epo for Epo oranti-Epo-bp antibodies for Epo-bp detection. For antibody detection,plates were coated with 200 μl of 1:10 diluted serum or plasma in PBS,pH 7.4 and incubated at room temperature for 30 min or stored at 4° C.overnight.

[0127] 2) Wells were washed 3 times with 200 μl/well PBST (0.05% Tween20 (Sigma Chemical Co., MO) in PBS).

[0128] 3) Nonspecific binding sites were blocked by adding 200 μl 1% BSAin PBST to each well and incubating for 30 min at room temperature.

[0129] 4) Wells were washed 3 times with 200 μl/well PBST.

[0130] 5) Streptavidin (2 μg/well in 200 μl PBST) labeled antibodies(Fab anti-Epo-antibody for Epo, Epo for Epo antibody, Fab anti-Epo-bpantibody for Epo-bp or Epo-bp for anti-Epo-bp antibody detection) wereadded to wells and samples were incubated at room temperature for 30min.

[0131] 6) Wells were washed 3 times with 200 μl/well PBST.

[0132] 7) To each well, 160 μl of O-Phenylene-Diamine Dihydrochloride(oPD) (Sigma Chemical Co., MO) in Phosphate-Citrate buffer, pH 6, wereadded and incubated for 30 min at room temperature.

[0133] 8) The reaction was stopped by adding 40 μl 5N NaOH and theabsorbance was measured at 405 nm.

[0134] oPD in phosphate citrate buffer was prepared as follows. For 100ml, 4.86 ml 0.5 M Citrate, 10.28 ml 0.5 M Na₂HPO₄, 2 ml oPD stock (10mg/ml) and up to 100 ml in dH₂O, pH 6.0. Immediately before use, 400 μlof 3% H₂O₂ were added.

[0135] The results of the EIA are presented in FIG. 11, in which opticaldensity (OD) of each measurement is presented as the mean±SE of 8-14individual samples (in duplicates). The OD measurements presented inFIG. 11 were calculated by subtracting the OD value of the blanks fromthe OD of each sample. As shown in FIG. 11, OD of Epo and Epo-bp inserum and plasma are similar to each other (OD₄₀₅: 0.308±0.026,0.289±0.022, 0.289±0.028 and 0.299±0.015 for serum Epo, serum Epo-bp,plasma-Epo and plasma-Epo-bp, respectively). The plasma level ofanti-Epo-bp antibody level was significantly lower than that of theother three categories: 0.058±0.008; 0.052±0.006; 0.054±0.013; and0.031±0.004 for serum anti-Epo, serum anti-Epo-bp, plasma anti-Epo andplasma anti-Epo-bp, respectively (p<0.025). Antibodies against Epo orEpo-bp in serum and Epo in plasma appeared to be similar. The Epo andEpo-bp values were converted with known Epo concentrations prepared ascontrols in the same plate to mU/ml. In serum 25.4±2.17 mU/ml of Epo and24.2±1.84 mU/ml of Epo-bp were present. In plasma, 24.2±2.35 mU/ml ofEpo and 25.0±1.26 mU/ml Epo-bp were present.

[0136] This assay is a simple method for measuring Epo and Epo-bp andprovides a more sensitive assay than measuring Epo and Epo-bp byradioimmunoassay (17.7±6.3 mU/ml) in terms of unit numbers and muchsmaller SE. Furthermore, the materials used in the preparation areenvironmentally friendly as compared with materials used in theconventional methods, such as radioactive or other toxic chemicals.

[0137] The foregoing detailed description has been provided for a betterunderstanding of the invention only and no unnecessary limitation shouldbe understood therefrom as some modifications will be apparent to thoseskilled in the art without deviating from the spirit and scope of theappended claims.

1 3 1 23 DNA Artificial sequence BamH1 linker followed by sequencecomplementary to coding sequence for amino acids 25-29 of human EpoRprotein 1 ttggatccgc gcccccgcct aac 23 2 22 DNA Artificial sequenceEcoR1 linker followed by sequence complementary to coding sequence for asegment of human EpoR protein 2 tgaattcggg gtccaggtcg ct 22 3 18 DNAHomo sapiens 3 ctggttccgc gtggatcc 18

What is claimed:
 1. A method for simultaneous measurement oferythropoietin (Epo) and Epo receptor in a biological sample, saidmethod comprising: a) providing a solid substrate having first andsecond antibodies attached thereto in different, discrete regions,wherein said first antibody has specific binding affinity for Epo andsaid second antibody has specific binding affinity for EpoR, and whereinsaid first and second antibodies are functional antibody fragments; b)contacting said solid substrate with said biological sample underconditions wherein Epo and EpoR in said biological sample becomes boundto said first and said second antibodies; and c) detecting the presence,absence, or amount of Epo and EpoR on said solid substrate.
 2. Themethod of claim 1, wherein said functional antibody fragments are Fabfragments.
 3. The method of claim 1, wherein said first and secondantibodies are polyclonal antibodies.
 4. The method of claim 1, whereinsaid detecting the presence, absence, or amount of Epo and EpoRcomprises contacting said solid substrate of part b) with third andfourth antibodies, wherein said third antibody has specific bindingaffinity for Epo and said fourth antibody has specific binding affinityfor EpoR, and wherein said third and fourth antibodies are detectablylabeled.
 5. The method of claim 4, wherein said third and fourthantibodies are polyclonal antibodies.
 6. The method of claim 1, whereinsaid detecting the presence, absence, or amount of Epo and EpoRcomprises: d) contacting said solid substrate of part b) with third andfourth antibodies, said third antibody having specific binding affinityfor Epo and said fourth antibody having specific binding affinity forEpoR; and e) contacting said solid substrate of part d) with fifthlabeled antibodies having specific binding affinity for said third andfourth antibodies.
 7. The method of claim 6, wherein said third andfourth antibodies are polyclonal antibodies.
 8. The method of claim 6,wherein said fifth labeled antibodies are labeled with an enzyme, asubstrate, or a fluorescent moiety.
 9. A kit for detecting Epo and EpoRin a biological sample, said kit comprising: (a) a solid substratehaving first and second antibodies attached thereto in different,discrete regions, wherein said first antibody has specific bindingaffinity for Epo and said second antibody has specific binding affinityfor EpoR, and wherein said first and second antibodies are functionalantibody fragments; (b) a first container having third antibodiesenclosed therein, wherein said third antibodies have specific bindingaffinity for Epo; and (c) a second container having fourth antibodiesenclosed therein, wherein said fourth antibodies have specific bindingaffinity for EpoR.
 10. The kit of claim 9, wherein said kit furthercomprises a third container having control antigen enclosed therein. 11.The kit of claim 9, wherein said kit further comprises a label orpackage insert indicating that Epo and EpoR can be simultaneouslydetected by contacting said solid substrate with said biological sampleunder conditions wherein any Epo or EpoR in said biological samplebecomes bound to said first and second antibodies and contacting saidsolid substrate with Epo or EpoR bound thereto with said third and saidfourth antibodies.
 12. The kit of claim 9, wherein said first and secondantibodies are polyclonal antibodies.
 13. The kit of claim 9, whereinsaid first and second antibodies are functional antibody fragments. 14.The kit of claim 9, wherein said third and fourth antibodies arepolyclonal antibodies.
 15. The kit of claim 9, wherein said solidsubstrate is a microtiter plate.
 16. A method for detecting thepresence, absence, or amount of EpoR on human blood progenitor cells,said method comprising: a) contacting a biological sample withantibodies having specific binding affinity for EpoR, wherein saidbiological sample contains human blood progenitor cells, and whereinsaid contacting occurs under conditions wherein antibodies becomes boundto EpoR on said human blood progenitor cells in said biological sample;and b) detecting the presence, absence, or amount of EpoR by identifyingsaid human blood progenitor cells having antibody bound thereto.
 17. Themethod of claim 16, wherein said biological sample is blood or bonemarrow cells.
 18. The method of claim 16, wherein said functionalantibody fragments are Fab fragments.
 19. The method of claim 16,wherein said human blood progenitor cells comprise megakaryocytes,erythroid progenitor cells, and myeloid progenitor cells.